Methods for Producing Polypeptidies in Protease-Deficient Mutants of Trichoderma

Active Publication Date: 2012-11-15
NOVOZYMES INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0060]An advantage of the present invention is elimination or reduction of one or more (several) enzyme activities, which may be detrimental to the production, downstream processing, e.g., recovery, and/or application of a particular polypeptide of interest
[0061]In the methods of the present invention, the parent Trichoderma strain may be any Trichoderma strain such as a wild-type Trichoderma strain or a mutant thereof. The parent Trichoderma strain may be Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride; or the alternative sexual form thereof, i.e., Hypocrea.
[0062]In another aspect, the parent Trichoderma strain is Trichoderma harzianum. In another aspect, the parent Trichoderma strain is Trichoderma koningii. In another aspect, the parent Trichoderma strain is Trichoderma longibrachiatum. In another aspect, the parent Trichoderma strain is Trichoderma reesei. In another aspect, the parent Trichoderma strain is Trichoderma viride.
[0063]In another aspect, the parent Trichoderma reesei strain is Trichoderma reesei RutC30. In another aspect, the parent Trichoderma reesei strain is Trichoderma reesei TV10. In another aspect, the parent Trichoderma reesei strain is a mutant of Trichoderma reesei. In another aspect, the parent Trichoderma reesei strain is a morphological mutant of Trichoderma reesei (see WO 97/26330).
[0064]The enzyme-deficient Trichoderma mutant

Problems solved by technology

The fermentation may not be optimal because of the production of biological substances, e.g., enzym

Method used

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  • Methods for Producing Polypeptidies in Protease-Deficient Mutants of Trichoderma
  • Methods for Producing Polypeptidies in Protease-Deficient Mutants of Trichoderma
  • Methods for Producing Polypeptidies in Protease-Deficient Mutants of Trichoderma

Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of Plasmid pDM156.2

[0174]A probe of a Neurospora crassa orotidine-5′-monophosphate decarboxylase (pyr-4) gene (SEQ ID NO: 7 for the DNA sequence and SEQ ID NO: 8 for the deduced amino acid sequence) was prepared by PCR incorporating digoxigenin-labeled deoxyuridine-triphosphate (dUTP) using the primers described below.

Primer (sense):(SEQ ID NO: 9)5′-GTCAGGAAACGCAGCCACAC-3′Primer (anti-sense):(SEQ ID NO: 10)5′-AGGCAGCCCTTGGACGACAT-3′

[0175]Plasmid pFB6 (Buxton et al, 1983, Molecular and General Genetics 190: 403-405) was digested with Hind III and the digestion purified by 1% agarose gel electrophoresis using 40 mM Tris base-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer. A 1.1 kb pyr-4 fragment was excised from the gel and extracted using a QIAQUICK® Gel Extraction Kit (QIAGEN Inc., Valencia Calif., USA) according to the manufacturer's suggested protocols.

[0176]The amplification reaction (50 μl) was composed of 1×Taq DNA Polymerase Buffer (New England Biolabs Inc.,...

example 2

Construction of Plasmid pEmY21

[0182]An E. coli hygromycin phosphotransferase (hpt) gene (SEQ ID NO: 11 for the DNA sequence and SEQ ID NO: 12 for the deduced amino acid sequence) was amplified from plasmid pPHTI (Cummings et al., 1999, Current Genetics 36: 371-382) using the following primers:

Forward primer:(SEQ ID NO: 13)5′-GGGttcgaaTTCATTTAAACGGCT-3′Reverse primer:(SEQ ID NO: 14)5′-GGGagcgctCAATATTCATCTCTC-3′

The restriction enzyme sites Bst BI (forward primer) and Eco 47111 (reverse primer) were engineered into the primers, represented by the underlined sequence, for cloning.

[0183]The PCR reaction (to amplify the hpt gene) was composed of 1×ThermoPol reaction buffer (New England Biolabs, Inc, Ipswich, Mass., USA), 200 μM dNTPs, 50 pmol of the forward and reverse primers, 100 pg of pPHT1, 1 unit of VENT® DNA polymerase (New England Biolabs Inc., Ipswich, Mass. USA), and sterile distilled water in a total volume of 100 μl. The amplification reaction was performed using a ROBOCYCLER®...

example 3

Construction of Plasmid pEmY23

[0191]The Fusarium venenatum pyrG coding sequence (2,678 bp) was excised from pDM156.2 (Example 1) by digestion with Eco RV and Stu I restriction endonucleases, and the remaining 4,398 bp vector was gel-purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's directions. The Sma I fragment of pEmY21 was isolated and gel-purified using a QIAQUICK® Gel Extraction Kit and the two gel-purified fragments were ligated together. They were screened for insert orientation, sequenced for the absence of errors, and one of the clones with the correct insert sequence was selected and designated pEmY23 (FIG. 3).

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Abstract

The present invention relates to mutants of a parent Trichoderma strain, comprising a polynucleotide encoding a polypeptide and one or more (several) genes selected from the group consisting of a first subtilisin-like serine protease gene, a first aspartic protease gene, a trypsin-like serine protease gene, a second subtilisin-like serine protease gene, and a second aspartic pro-tease gene, wherein the one or more (several) genes are modified rendering the mutant strain deficient in the production of one or more (several) enzymes selected from the group consisting of a first subtilisin-like serine protease, a first aspartic protease, a trypsin-like serine protease, a second subtilisin-like serine protease, and a second aspartic protease, respectively, compared to the parent Trichoderma strain when cultivated under identical conditions. The present invention also relates to methods of producing a polypeptide in such mutants and methods for producing such mutants.

Description

REFERENCE TO A SEQUENCE LISTING This application contains a Sequence Listing filed electronically by EFS, which is incorporated herein by reference.BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The present invention relates to methods of producing polypeptides in enzyme-deficient Trichoderma mutant strains, the enzyme-deficient Trichoderma mutant strains, and methods of obtaining the enzyme-deficient Trichoderma mutant strains.[0003]2. Description of the Related Art[0004]Trichoderma has been shown to be useful as a host cell for the recombinant production of polypeptides having biological activity (WO 96 / 00787, WO 97 / 26330). Trichoderma hosts with the desirable traits of increased protein expression and secretion may not necessarily have the most desirable characteristics for successful fermentation. The fermentation may not be optimal because of the production of biological substances, e.g., enzymes, detrimental to the production, recovery, or application of a par...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/80C12P21/00
CPCC12N9/58C12P21/02C12N15/80C12N15/04C12P21/00
Inventor MAIYURAN, SUCHINDRAJANG, ABIGAILBROWN, KIMBERLYMERINO, SANDRASHASKY, JEFFREYABBATE, ERIC
Owner NOVOZYMES INC
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